Project Summary/ Abstract Lung cancer is the leading cause of cancer-related deaths in the US and disproportionally affects Veterans. Advances in non-small-cell lung cancer over the past decade have resulted in new molecularly targeted therapies with minimal toxic effects and dramatic clinical benefits. However, despite these progresses, overall five-year survival remains at approximately 16%, partly due to low responsive rate to targeted therapy, development of acquired resistance, difficulty of targeting certain proteins such as mutant K-RAS, and a large subset with undefined genetic alterations. Therefore, new molecular targets are needed for lung cancer subtypes that are currently refractory to available treatments. EphA2 is such a promising target. EphA2 is overexpressed in NSCLC, and high levels of EphA2 correlate with smoking, brain metastasis, disease relapse, and poor patient survival. A gain-of- function EphA2 mutation has also been identified in tumor specimens, suggesting an oncogenic role of EphA2 in lung cancer. Indeed, we discovered that knockdown of EphA2 in a large numbers of human lung cancer cell lines inhibited tumor cell viability, most dramatically affecting those bearing mutant K-RAS or carrying EGFR mutation that developed acquired resistance to tyrosine kinase inhibitors (TKI). To support this notion, an EphA2 small molecule kinase inhibitor suppressed cell viability in vitro and induced tumor regression in K-RAS mutant human lung cancer xenografts. Based on these preliminary data, the overall goal of this VA Merit renewal is to determine the efficacy of targeting EphA2 in lung cancer subtypes that are refractory to current targeted treatment, to elucidate molecular basis for EphA2 function in tumor, and to test small molecule EphA2 kinase inhibitors for cancer therapeutics. We will first to investigate the effects of EphA2 deficiency on lung cancer development and progression in vivo in transgenic K-RAS G12D and TKI-resistant EGFR L858R+T790M lung cancer models. To elucidate EphA2 receptor downstream signaling, we will focus on the JNK/c-Jun pathway in regulating tumor cell viability and tumor stem-like cell function. Finally, we will test the efficacy of selective small molecule inhibitors of EphA2 receptor. Success of this project will not only generate novel insights into the molecular basis whereby EphA2 RTK regulates tumor cell viability, but also provide novel EphA2- selective inhibitors for treatment of lung cancer subtypes that are refractory to current targeted therapies, such as K-RAS mutant and drug-resistant EGFR mutant lung cancers.